The objective of this research is to understand the mechanism by which the initial interaction of insulin with the receptor at the cell surface results in the generation of an intracellular signal or message that stimulates cellular metabolism. I have developed a photoaffinity labeling technique (involving the use of arylazide heterobifunctional crosslinking reagents) for specific covalent radiolabeling of cell surface receptors for polypeptide hormones. Cells containing an 'in situ' affinity radiolabeled insulin receptor will be used for following the dynamic and migratory behavior of the receptor after binding to insulin. The photoaffinity labeling technique will be further exploited to monitor purification of the receptor which will be used as an immunogen for generation of hybridomas secreting specific antireceptor antibodies. The antibody will be used as a tool to test the physiological significance of receptor internalization or endocytosis in the biological action of insulin. A cytobiochemical assay will be developed for the intracellular second messenger induced by insulin. Cytoplasmic extracts from insulin-stimulated cells will be introduced into unstimulated cells by microinjection or encapsulation, and the effects on glucose oxidation, DNA and RNA synthesis will be measured. Anti-receptor antibody will be used to deplete the assay system of receptor/processed receptor, and the biological effects of this depletion will be examined. These detailed cytobiochemical studies on the receptor and the second messenger will aid greatly in achieving my long term goals: complete definition of the molecular events in insulin action, and assessment of the defects in receptor activating mechanism in insulin-resistant diabetes.